Fig 1: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.